Academic literature on the topic 'PCR diagnostic'

Polymerase chain reaction (PCR)-based diagnostics is currently established as a gold standard for the detection of microorganisms. The features of PCR include rapid amplification of DNA and RNA as well as high sensitivity and specificity. In contrast to diagnostic microbiology, PCR diagnostics does not require preliminary culture of the microorganisms for their identification, reducing both time and costs of the diagnostic procedure. The lecture discusses the molecular basis behind the modern technical solutions for the PCR diagnostics of human infectious diseases including multiplex and reverse transcription PCR. We describe the principles of qualitative and quantitative PCR-based detection of pathogens in biological samples and provide the examples of PCR application for solving specific diagnostic scenarios. The lecture is primarily designed for students of biomedical specialties and healthcare professionals using molecular genetic techniques in their practice.

‘Candidatus Liberibacter solanacearum’ is an economically important pathogen in the Americas, New Zealand and Europe. The primary objective of this review is to systematically investigate the polymerase chain reaction (PCR)-based methods used for its detection in plant samples. Several databases were searched from the inception of the relevant literature up to August 2018. This review identified 53 studies that met all the inclusion criteria. The performance of the different methods was also compared, however due to data heterogeneity and insufficient evidence on the sensitivity of all assays used, a meta-analysis of the data was not possible. Nonetheless, the review indicates that the rtPCR designed to the 16S ribosomal RNA gene can be routinely employed as a fast, cost-effective, and reliable detection technique in diagnostic laboratories.

La leishmaniose viscerale (lv) s'etend depuis quelques annees en plusieurs regions du monde a la suite de changements epidemiologiques tels que l'urbanisation ou les mouvements de populations. Dans le meme temps, des coinfections leishmania/virus de l'immunodeficience humaine (vih) sont de plus en plus souvent rapportees, principalement sur le pourtour du bassin mediterraneen. L'amelioration du diagnostic biologique de la lv et l'evaluation de nouveaux traitements antileishmaniens ont constitue les objectifs de notre travail. Une technique de pcr pour le diagnostic des leishmanioses et plus particulierement pour le diagnostic de l. Infantum dans le cadre de l'association au vih a ete developpee. Des mesures physiques (locaux et materiels adaptes) et chimiques (decontamination enzymatique par emploi de l'uracyl-d-glycosylase) ont ete prises de maniere a eviter les faux positifs. Un controle interne de type heterologue, permettant la detection des inhibiteurs de la reaction, a ete developpe de maniere a eviter les faux negatifs. La technique a ete optimisee avec detection des produits d'amplification par technique elisa ce qui a permis d'augmenter la sensibilite et de controler la specificite de l'amplification. La pcr diagnostique a ete evaluee par comparaison avec les techniques classiques de diagnostic de leishmaniose : - aucun faux positif par pcr n'a ete observe pour les 77 sujets non atteints de leishmaniose au moment du prelevement. Ces sujets n'ont pas declare de leishmaniose dans les 12 mois ayant suivi le prelevement. - les prelevements positifs a l'examen direct et/ou a la culture ont toujours ete positifs par pcr a l'exception d'un cas. - la pcr a ete positive pour 6 prelevements provenant de patients leishmaniens connus alors que examens directs et cultures etaient negatifs. La vectorisation consiste a masquer les molecules actives au sein d'une particule capable de les conduire jusqu'a leur cible et de ne les liberer qu'a ce moment. Les phenomenes de toxicite sont donc normalement diminues, accroissant l'index therapeutique de la molecule vectorisee. Les principaux vecteurs synthetises au cours de ce travail ont ete des nanoparticules de polymethacrylate, des nanoparticules d'acide poly (d,l-lactique) et des liposomes. L'activite antileishmanienne des nanoparticules chargees par la pentamidine et des liposomes charges par l'amphotericine b (amb) a ete evaluee au moyen d'un modele original associant la souris balb/c et l. Infantum. La plus forte augmentation d'activite antileishmanienne apportee par la vectorisation a ete observee pour les nanoparticules de polymethacrylate chargees par la pentamidine (activite multipliee par 6). La faible biodegradabilite de ces nanoparticules constitue cependant un handicap par rapport aux autres vecteurs evalues dans ce travail. Les nanoparticules d'acide (d,l) lactique, liant la pentamidine de maniere plus labile, ont multiplie l'activite de la pentamidine par 3. L'ultrastructure des leishmanies observee apres traitement par la pentamidine chargee sur des nanoparticules de polymethacrylate n'a pas montre d'alterations specifiques ou plus importantes que celles observees avec la pentamidine libre.

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

La tuberculose continue d’être une cause majeure de morbidité et de mortalité dans le monde, principalement dans les pays en voie de développement, bien qu’elle soit une maladie curable. Le diagnostic rapide et précis de la TB active est essentiel pour l’initiation rapide du traitement et le contrôle de la maladie. Le développement de nouveaux tests rapides de diagnostic de TB active représente un véritable challenge pour l’optimisation du diagnostic.L’objectif principal de nos travaux de thèse était de développer et d’évaluer des approches de PCR en temps réel ciblant la séquence d’insertion IS6110 pour la détection de l’ADN de MTB dans les expectorations.Nous avons tout d’abord développé une PCR en temps réel ciblant la séquence répétée IS6110 pour la quantification de l’ADN de MTB. L’évaluation des étapes d’optimisation de la sensibilité de la PCR IS6110 a permis de préciser les performances analytiques et le gain de sensibilité comparativement à une PCR ciblant le gène unique senX3. Au terme d’une comparaison de six protocoles de lyse/ extraction la méthode Chelex® s’est avérée être la plus efficace dans la récupération de l’ADN. La performance diagnostique de la PCR optimisée a été évaluée et comparée avec la PCR automatisée Xpert MTB/RIF sur un panel de 62 échantillons respiratoires.Dans un deuxième temps, nous avons comparé la performance diagnostique de la PCR IS6110 optimisée, le test Xpert MTB/RIF et la version ultrasensible récemment commercialisée du test PCR leader Xpert MTB/RIF Ultra pour la détection de l’ADN de MTB dans des expectorations ayant une faible charge bacillaire.Enfin, à partir de 203 LCR collectés dans le cadre du diagnostic de méningites aseptiques au Burkina Faso, nous avons évalué la performance de la PCR en temps réel multiplexe (IS6110, HSV1, HSV2) combinée à l’extraction par la méthode Chelex® pour la détection de l’ADN de MTB et d’Herpès
TTuberculosis continues to be a major cause of morbidity and mortality worldwide, mainly in developing countries, despite being a curable disease. The rapid and accurate diagnosis of active TB is essential for rapid initiation of treatment and disease control. The development of new rapid diagnostic tests for active TB represents a real challenge for the optimization of the diagnosis.The main objective of our thesis work was to develop and evaluate real-time PCR approaches targeting the IS6110 insertion sequence for the detection of sputum MTB DNA. We first developed a real-time PCR targeting the IS6110 repeat sequence for the quantification of MTB DNA. The evaluation of the sensitivity optimization steps of the IS6110 PCR made it possible to specify the analytical performances and the sensitivity gain compared to a PCR targeting the single gene senX3. After a comparison of six lysis / extraction protocols, the Chelex® method proved to be the most efficient in the recovery of DNA. The diagnostic performance of optimized PCR was evaluated and compared with automated Xpert MTB / RIF PCR on a panel of 62 respiratory specimens.In a second step, we compared the diagnostic performance of the optimized IS6110 PCR, the Xpert MTB / RIF test and the highly marketed ultra-sensitive version of the Xpert MTB / RIF Ultra leader PCR assay for the detection of sputum MTB DNA. having a low bacillary load.Finally, from 203 LCR collected in the context of the diagnosis of aseptic meningitis in Burkina Faso, we evaluated the performance of the multiplexed real-time PCR (IS6110, HSV1, HSV2) combined with extraction by the Chelex® method for the detection of MTB and Herpes DNA

La toxoplasmose est une parasitose ubiquitaire occupant une large place en médecine humaine et vétérinaire. Cette affection parasitaire très fréquente en France est le plus souvent bénigne voire asymptomatique. Pourtant, elle reste redoutable en situation de transmission congénitale. De même, sa survenue chez les malades immunodéprimés a radicalement changé la conception de cette maladie. Pour compenser les insuffisances du sérodiagnostic et du diagnostic parasitologique, nous avons mis au point un diagnostic permettant un dépistage très précoce face à la suspicion d'une toxoplasmose congénitale ou cérébrale. Ce diagnostic repose sur l'amplification spécifique d'un gène répété 35 fois de Toxoplasma gondii (gène B1) associé à un système de révélation par chimioluminescence. A partir de prélèvements comparables aux liquides amniotiques humains, nous sommes capables de détecter spécifiquement un tachyzoïte. Nous avons également mis au point un modèle de PCR quantitative sur ADN utilisant un standard interne compétitif et spécifique des gènes SAGl, B1 et ARN 18S. Ce modèle est applicable à tout modèle d'étude expérimental où la quantification d'une charge parasitaire est nécessaire. Nos premiers essais ont porté sur la quantification d'une charge parasitaire cérébrale (kyste, souche Prugniaud) dans un modèle murin (BALB/C et C57 BI/6) d'encéphalite toxoplasmique. Afin de documenter une éventuelle corrélation entre l'expression des gènes de granules denses et la virulence des différentes souches du parasite, nous avons élaboré l'ensemble des outils techniques d'un modèle de RT-PCR quantitative pour le gène GRA2 (gène reconnu être impliqué dans le pouvoir pathogène). Ce système utilise une molécule standard interne compétitive, différente de 4 paires de bases comparée à la cible génomique. Les produits PCR marqués à la fluorescéine par nested-PCR, seront quantifiés à l'aide d'un analyseur automatique d'ADN. Les variations de rendement d'amplification seront quant à elles, corrigées par la quantification d'un gène rapporteur (bêta-tubuline).

Les tests moléculaires sont fréquemment demandés pour le diagnostic et le suivi des maladies infectieuses dans les pays développés. Cependant, sa disponibilité reste limitée dans les pays à ressources limitées dû à des contraintes de coûts, des technologies et des ressources humaines. Dans les régions éloignées, un accès limité aux installations de laboratoire constitue également un problème majeur. Le développement de méthodes de PCR sur plateformes ouvertes dans des laboratoires de référence tels que le Centre MURAZ au Burkina-Faso et l'utilisation d’échantillons de sang total capillaires sur DBS peuvent faciliter l'accès aux tests moléculaires.Selon la directive de l'OMS, la quantification de l'ARN du VIH à l'aide de DBS peut être une alternative dans les contextes d’accès difficile au laboratoire. Une préoccupation majeure est la spécificité sous-optimale de l’utilisation des DBS en raison de l'interférence de l'ADN du VIH archivé dans des cellules infectées sur la charge virale ARN du VIH. Dans une première étude, nous avons déterminé le niveau d'ADN du VIH-1 qui entravait la fiabilité de la quantification de l'ARN du VIH-1 sur des DBS. Une détection d'ARN du VIH-1 faussement positif (22/62, 35%) a été associée à des niveaux élevés d'ADN de VIH-1. Nos résultats indiquent que la spécificité des tests d'ARN du VIH-1 sur les DBS devrait être évaluée suivant les protocoles du fabricant sur les échantillons avec des niveaux d'ADN de VIH-1 de ≥1 000 copies / 106 cellules mononuclées du sang périphérique.Outre les infections fréquemment diagnostiquées, il est urgent d'intensifier la recherche d’infections négligées comme la leptospirose. Dans une seconde étude, nous avons exploré la leptospirose par des tests sérologiques et moléculaires comme cause négligée de maladie chez les patients atteints d’ictère fébrile au Burkina Faso. Les résultats ont montré pour la première fois que la leptospirose est une cause insoupçonnée de maladie fébrile aiguë dans ce pays semi-aride.Dans la dernière partie du doctorat, nous avons développé un test de PCR multiplex pour le diagnostic de la méningite à herpès simplex virus (HSV) et à Mycobacterium tuberculosis (MTB) chez des patients suspects de méningite aseptique. Cette qPCR qui a permis de tester dans un seul essai HSV 1/2 et MTB était très spécifique, sensible et reproductible. La concentration d'ADN la plus faible donnant 100% de signal de détection a été estimée à 2,12 copies / μl pour HSV1, 1,76 pour HSV2 et 2,15 copies / μl pour MTB. Parmi les 202 échantillons de LCR inclus dans cette étude, 5 (2,46%) étaient positifs: 2 (0,99%) pour HSV et 3 (1,47%) pour MTB. Ce test peut être particulièrement utile dans les cas de méningite / encéphalite avec un faible nombre de globules blancs dans le CSF.Notre projet a montré l'importance de la mise en œuvre de nouvelles méthodes moléculaires pour fournir des données préliminaires sur le fardeau des maladies infectieuses, y compris la leptospirose, la tuberculose et la méningite à HSV au Burkina Faso. Le DBS est un spécimen alternatif qui facilite l'accès aux tests moléculaires mais nécessite des études de validation. Les approches syndromiques doivent être testées et mises en œuvre dans les pays à ressources limitées en se basant sur l'expertise locale et la mise en œuvre de méthodes moléculaires dans les laboratoires de référence
Molecular assays are frequently requested for the diagnosis and monitoring of infectious diseases. While nucleic acid testing is the standard of care in developed countries, its availability remains limited and constrained by cost, technologies, and human resources in many settings, including West Africa. In remote areas, limited access to laboratory facilities is also a main issue. The development of PCR methods on open polyvalent platform in reference laboratories such as the Centre Muraz in Burkina-Faso and the use of capillary whole blood collected on DBS specimens can facilitate access to nucleic acid testing.According to WHO guideline HIV-RNA quantification using DBS can be considered in settings where there is a lack of access to sites or nearby laboratory facilities for nucleic acid test. A major concern is the suboptimal lower specificity of DBS due to the interference of HIV-DNA copies archived in infected cells with HIV-RNA copies. In the first study we determined the HIV-1 DNA level that interfered with the reliability of HIV-1 RNA quantification on DBS specimens used for therapeutic monitoring (1). False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Our results indicate that the specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/106 peripheral blood mononuclear cells.Beside infections frequently tested by nucleic acid tests there is an urgent need to scale up assay for neglected infection such leptospirosis. In the second study we explored leptospirosis by serological and molecular testing as a neglected cause of disease among patients with febrile icteric illness in Burkina Faso. The results showed for the first time that leptospirosis is an unsuspected cause of acute febrile illness in this semi-arid country.In the last part of the PhD, we developed a multiplex PCR assay for the diagnosis of tuberculosis and Herpes Simplex (HSV) meningitis among patients with suspected aseptic meningitis. This qPCR which allowed to test in a single run HSV 1/2 and M. tuberculosis was highly specific, sensitive and reproducible. The lowest DNA concentration giving 100% detection signal was estimated at 2.12 copies/µl for HSV1, 1.76 for HSV2 and 2.15 copies/µl for M. tuberculosis. Of the 202 CSF specimens included in this study, 5 (2.46%) were tested positive: 2 (0.99%) for HSV and 3 (1.47%) for M. tuberculosis. This assay may be especially useful in cases of meningitis/encephalitis with a low number of white blood cells count in the CSF.Our project stresses the importance of the implementation of news molecular methods to provide preliminary data about the burden of infectious diseases including leptospirosis, tuberculosis and HSV meningitis in Burkina Faso. DBS is an alternative specimen that facilitates access to nucleic acid tests but requires validation studies. Syndromic approach need to be tested and implemented in West Africa and should be based on local expertise and implementation of molecular methods in reference laboratories

BACKGROUND: Diagnostic molecular testing in colorectal cancer (CRC) offers a number of benefits including predicting prognosis, directing targeted therapies and screening for hereditary cancer syndromes. Molecular testing however is expensive, requires specialist facilities and staff and is time consuming, limiting its widespread availability. The Idylla System is an automated testing platform that could overcome these issues. AIMS: To appraise the suitability of the Idylla System for use in clinical practice by evaluating the system’s accuracy and financial impact. HYPOTHESIS: The Idylla System has high accuracy for detecting mutations in BRAF, KRAS and NRAS genes in CRC resection tissue and is a cost-effective alternative to current testing platforms. METHODS: Ethical approval was granted by Oxfordshire Research and Ethics Committee A (reference: 04/Q1604/21). Diagnostic accuracy was determined for the Idylla System in detecting BRAF and KRAS mutations with a comparison against conventional polymerase chain reaction (PCR). Further validations were also performed for BRAF, KRAS and NRAS mutation testing against NGS and IHC methods. An audit of the molecular diagnostics workload was carried out and a cost-analysis performed. RESULTS: The Idylla system had a sensitivity of 100.0% (95% CI: 88.3% to 100.0%) and a specificity of up to 100.0% (95% CI: 94.7% to 100.0%) for detecting BRAF mutations and a sensitivity of 100.0% (95% CI: 79.6% to 100.0%) and a specificity of up to 92.9% (95% CI: 68.5% to 98.7%) for detecting KRAS Mutations. There was 100% concordance for NRAS testing. A cost-analysis estimated that the Idylla System could save from around £12,000 to anywhere up to £40,000 per year in some centres. CONCLUSIONS: The results support the hypothesis that the Idylla System is an accurate system for detecting relevant mutations in CRC and demonstrate the system to be cost-effective. The Idylla system is therefore suitable for use in routine clinical practice.

Cette thèse présentait comme objectif mettre en évidence le potentiel apport de l’immuno-PCR dans le diagnostic des maladies infectieuses à travers 3 exemples. L’application de l’iPCR dans le diagnostic précoce de la fièvre Q aiguë via la détection des IgM Phase II anti Coxiella burnetii a permis de détecter 90% des sérums prélevés dans les 2 premières semaines après l’apparition des symptômes contre 55% détectés par PCR, 38% par ELISA et 35% par l’IF la technique de référence. De plus une spécificité de 92% a été retrouvée par iPCR, 100% par PCR et l’IF et 90% par ELISA. L’application de l’iPCR à la fièvre Q aiguë constitue un exemple d’application de la technique plus globalement pour tous types d’infections aiguës. D’autre part, dans le cadre de la mise au point d’un modèle expérimental murin d’infection à Tropheryma whipplei, l’iPCR a servi à mesurer la réponse immunitaire mucosale via la détection des IgA anti T. whipplei dans les selles de souris. Les résultats obtenus ont permis de confirmer le rôle de la bactérie comme agent de gastroentérite via entre autre la détection d’IgA anti T. whipplei dans les selles lorsque des atteintes intestinales étaient provoquées. Enfin l’’utilisation de l’iPCR pour la détection de l’antigène Y. pestis dans des dents anciennes a permis de confirmer Y. pestis comme agent étiologique de la peste noire dans 5 charniers à travers la France et l’Italie. Une sensibilité de 41% a été retrouvée par iPCR contre 32% par PCR et 10% par ELISA. Nos résultats suggèrent que la détection des antigènes et la détection de l’ADN du pathogène semblent être 2 approches complémentaires permettant de confirmer le rôle de Y. pestis dans les différentes pandémies de peste et de mettre fin aux controverses suscitées par la seule utilisation des techniques de biologie moléculaire.Globalement, les résultats que nous avons obtenus au cours de cette thèse démontrent le potentiel énorme de l’iPCR comme technique de détection des anticorps et d’antigènes dans le domaine des maladies infectieuses tant au niveau de la sensibilité de détection qu’au niveau de son adaptabilité à différentes applications
The objective of this thesis was to highlight the potential contribution of immuno-PCR in the diagnosis of infectious disease through 3 examples of infection. The iPCR was adapted for the early diagnosis of acute Q fever by the detection of IgM anti phase II Coxiella burnetii in patient’s sera. The results that we obtained show that iPCR could allow an early diagnosis of acute Q fever since 90% of early sera of patients with acute Q fever collected during the 2 first weeks after the onset of symptoms are detected against 55% by PCR, 38% by ELISA and 35% by IFA the gold standard. In addition, a specificity of 92% was found by iPCR, 90% by ELISA and 100% by PCR and IF. Application of iPCR to the diagnosis of acute Q fever is an example of application of the technique more generally for all types of acute infections. In a second time, the use of iPCR for the detection of Yersinia pestis antigen in ancient teeth allowed a confirmation of its role as the etiologic agent of plague in five mass graves across France and Italy. A sensitivity of 41% was recovered by IPCR against 32% by PCR and 10% by ELISA. Our results suggest that antigen and DNA detection of pathogen in ancient samples are 2 complementary approaches allowing the confirmation of the role of Y. pestis in different plague pandemic. Finally, as part of the development of an experimental murine infection with Tropheryma whippleii, the iPCR was used to measure the mucosal immune response via the detection of IgA anti T. whippleii in mouse stools. The results have confirmed the role of the bacterium as an agent of gastroenteritis via the detection of IgA anti T. whipleii in the stool when intestinal damages were caused.Overall, the results that we obtained during my thesis demonstrate the enormous potential of iPCR as a diagnostic tool of infectious disease by the ultrasensitive detection of antigens and antibodies

"Diagnosing infections with SARS-CoV-2 is still of great interest due to the health and economic impact of COVID pandemic. The 4th wave of the COVID-19 pandemic is expected and is considered to be stronger and faster due to the dominance of Delta variant which is highly contagious [1]. SARS-CoV-2 also known as 2019-nCoV is one of the three coronaviruses (together with SARS-CoV or SARS-CoV1/Severe acute respiratory syndrome coronavirus), MERS-CoV /Middle East Respiratory Syndrome coronavirus) which can cause severe respiratory tract infections in humans [2]. Early diagnosis in COVID 19 infection is the key for preventing infection transmission in collectivity and proper medical care for the ill patients. Gold standard for diagnosing SARS-Co-V-2 infection according to WHO recommendation is using nucleic acid amplification tests (NAAT)/ reverse transcription polymerase chain reaction (RT-PCR). The search is on to develop reliable but less expensive and faster diagnostic tests that detect antigens specific for SARS-CoV-2 infection. Antigen-detection diagnostic tests are designed to directly detect SARSCoV-2 proteins produced by replicating virus in respiratory secretions so-called rapid diagnostic tests, or RDTs. The diagnostic development landscape is dynamic, with nearly a hundred companies developing or manufacturing rapid tests for SARS-CoV-2 antigen detection [3]. In the last 3 months our hospital introduced the antigen test or Rapid diagnostic tests (RDT) which detects the presence of viral proteins (antigens) expressed by the COVID-19 virus in a sample from the respiratory tract of a person. All RDT were confirmed next day with a RT-PCR. The number of positive cases detected during 3 months in our laboratory was 425. There were 326 positive tests in April, 106 positive tests in May and 7 positive tests in June. Compared with the number of positive tests in the 1st semester of 2021, the positive tests have significantly declined."

In resource-limited settings, it is impractical to get access to a diagnostic laboratory having sophisticated instruments, and it is desirable to use disposable point-of-care diagnostic chips that do not require liquid handling or pumping instruments for sample distribution among an array of reactors. In addition to the pump-less sample loading method, the challenge to seal an array of reactors without the use of microvalves or mechanical parts still persists. Implementation of microvalve array adds complexity to the chip fabrication and operation processes, and also reduces the space on the microchip. In this paper, we report the development of a high-throughput quantitative PCR chip platform for parallel analyses of multiple gene targets. The PCR mixture distribution among an array of 80 microreactors and subsequent isolation of the reactors were solely realized by a two-step surface tension-based microfluidic scheme, which eliminates the use of pumps, valves and liquid handling instruments. Confinement of the PCR mixture inside the micro reactors was achieved by implementing hybrid flow-restriction passive valves. The microreactors were isolated from each other by the flow of a curable liquid sealant delivered through microchannels by capillary action. We also investigated the effect of detergents that are present in most commercial PCR buffers. Presence of detergents makes the PCR buffer much more wetting on the passive capillary valve surface and this imposes another challenge to the design of the conventional hydrophobic patch valves which has been successfully used for deionized water. We demonstrated a successful capillary valve array with a common air venting channel having a hydrophobic surface for restricting the flow of PCR buffer containing surfactant. The interconnected microreactor array was fabricated on a glass chip substrate with approximate volume of 250 nl microreactor volume for PCR. A different set of PCR primers were preloaded into different microreactor on the PCR array chip for simultaneous amplification of multiple genes. Fluorescent signals from all the microreactors were simultaneously detected at every PCR thermal cycle using EvaGreen fluorescent dye on an in-house real-time PCR instrument. The capability of the scalable PCR array chip was demonstrated by amplifying a fragment of uidA gene for beta-glucuronidase of E. coli genome. Key technical issues related to chip operation such as PCR inhibition on the acid-washed glass substrate, and PCR compatibility of the sealant in both the uncured and cured states have been addressed.

 Evidence suggests that the majority of adults develop detectable levels of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies following infection with SARS-CoV-2 (moderate strength of evidence* [SoE]).  IgM levels peak approximately 20 days after symptom onset or RT-PCR diagnosis and subsequently decline. IgG levels peak approximately 25 days after symptom onset or RT-PCR diagnosis and may remain detectable for at least 120 days (moderate SoE*).  Almost all adults develop neutralizing antibodies in response to SARS-CoV-2 infection, and these antibodies may remain detectable for at least 152 days (low SoE*).  A small percentage of people do not develop antibodies in response to SARS-CoV-2 infection for reasons that are largely unclear but may be related to less severe disease or absence of symptoms.  Antibody prevalence does not appear to vary by age or sex, but older age may be associated with higher antibody levels (low SoE*). Non-White race may be associated with higher antibody prevalence and levels (low SoE*). COVID-19 severity and presence of symptoms may also be associated with higher antibody prevalence or levels (low SoE*). More evidence is needed to draw stronger conclusions regarding how the antibody response varies by patient characteristics and disease factors.  Studies to date have not established the relationship between the development of antibodies after RT-PCR-diagnosed SARS-CoV-2 infection and the risk of reinfection. Studies based on index serologic testing suggest that the presence of antibodies is associated with a lower risk of a subsequent positive SARS-CoV-2 RT-PCR test.

Abstract: Multiple studies have found that psychiatric disorders, like mood disorders and substance use disorders, are highly comorbid among adults with either disorder. Integrated treatment refers to the treatment of two or more conditions and the use of multiple therapies such as the combination of psychotherapy and pharmacotherapy. Integrated therapy for comorbidity per numerous studies has consistently been superior to the treatment of individual disorders separately. The purpose of this QI project was to identify the effectiveness of Cognitive Behavioral Therapy (CBT) instead of current treatment as usual for treating Substance Use Disorder (SUD) or mental health diagnosis independently. It is a retrospective chart review. The review examines CBT's efficacy for engaging individuals with co-occurring mood and substance u se disorders in treatment by enhancing adherence and preventing disengagement and relapse. Methods: Forty adults aged 26-55 with a DSM-IV diagnosis of a mood disorder of Major Depressive Disorder and/or anxiety and concurrent substance use disorder (at least weekly use in the past month). Participants received 12 sessions of individual integrated CBT treatment delivered with case management over a 12-week period. Results: The intervention was associated with significant improvements in mood disorder, substance use, and coping skills at 4, 8, and 12 weeks post-treatment. Conclusions: These results provide some evidence for the effectiveness of the integrated CBT intervention in individuals with co-occurring disorders. Of note, all psychotherapies are efficacious; however, it would be more advantageous to develop a standardized CBT that identifies variables that facilitate treatment outcomes specifically to comorbid disorders of substance use and mood disorders. It is concluded that there is potentially more to be gained from further studies using randomized controlled designs to determine its efficacy.

A cadeia mineral da Vale ao longo do Corredor Norte, localizado no leste da Amazônia, entre os estados do Pará e Maranhão, ocorre a céu aberto e está frequentemente exposta aos fenômenos atmosféricos indutores de mau tempo, como tempestades, incidência de raios, rajadas de vento, etc. Neste sentido, o presente relatório técnico teve como objetivo evidenciar os impactos das condições atmosféricas ao longo da cadeia mineral, com ênfase nos sítios de Carajás e no Terminal Marítimo Ponta da Madeira (TMPM). A abordagem foi conduzida com base na análise de dados meteorológicos e na percepção de empregados da Vale alocados em ambos os sítios. Os resultados demonstraram que o Corredor Norte da Vale é influenciado por sistemas atmosféricos, que atuam em diferentes períodos do ano conforme a região. A climatologia regional da precipitação demonstrou uma sazonalidade marcante, com períodos chuvosos e secos distintos entre Carajás e o TMPM, característica que é considerada no planejamento logístico e operacional da Vale, como constatado nas entrevistas com alguns empregados da Vale. Os resultados capturados em tais entrevistas evidenciaram que as condições atmosféricas estão diretamente associadas a atividades da Vale, como citado por gerências operacionais e manutenção, por exemplo. Além disso, a percepção do entrevistado da gerência de segurança ocupacional, monitorar e prever a eventos meteorológicos vai muito além da questão da produção, mas principalmente no ato de salvaguardar a vida dos empregados da Vale, bem como das comunidades que residem em áreas de mineração. Particularmente no TMPM, a criação de um Comitê de Umidade para a mitigação dos impactos atmosféricos na cadeia mineral, como foco em ações para os próximos períodos chuvosos, reforça que a Vale tem dedicado atenção na redução as incertezas em relação aos eventos meteorológicos danosos às suas operações, bem como tem investido em tecnologias aplicadas ao monitoramento e previsão de tempo. Os resultados encontrados no presente relatório técnico corroboram com o uso de tecnologias em meteorologia, capazes de diagnosticar o estado momentâneo da atmosfera, de modo que as informações relevantes sejam disponibilizadas no formato de alertas aos usuários interessados.

Many students struggle with mathematics in late elementary school, particularly on the topic of fractions. In a best evidence syntheses of research on increasing achievement in elementary school mathematics, Pelligrini et al. (2018) highlighted tutoring as a way to help students. Online tutoring is attractive because costs may be lower and logistics easier than with face-to-face tutoring. Cignition developed an approach that combines online 1:1 tutoring with a fractions game, called FogStone Isle. The game provides students with additional learning opportunities and provides tutors with information that they can use to plan tutoring sessions. A randomized controlled trial investigated the research question: Do students who participate in online tutoring and a related mathematical game learn more about fractions than students who only have access to the game? Participants were 144 students from four schools, all serving low-income students with low prior mathematics achievement. In the Treatment condition, students received 20-25 minute tutoring sessions twice per week for an average of 18 sessions and also played the FogStone Isle game. In the Control condition, students had access to the game, but did not play it often. Control students did not receive tutoring. Students were randomly assigned to condition after being matched on pre-test scores. The same diagnostic assessment was used as a pre-test and as a post-test. The planned analysis looked for differences in gain scores ( post-test minus pre-test scores) between conditions. We conducted a t-test on the aggregate gain scores, comparing conditions; the results were statistically significant (t = 4.0545, df = 132.66, p-value < .001). To determine an effect size, we treated each site as a study in a meta-analysis. Using gain scores, the effect size was g=+.66. A more sophisticated treatment of the pooled standard deviation resulted in a corrected effect size of g=.46 with a 95% confidence interval of [+.23,+.70]. Students who received online tutoring and played the related Fog Stone Isle game learned more; our research found the approach to be efficacious. The Pelligrini et al. (2018) meta-analysis of elementary math tutoring programs found g = .26 and was based largely on face-to-face tutoring studies. Thus, this study compares favorably to prior research on face-to-face mathematics tutoring with elementary students. Limitations are discussed; in particular, this is an initial study of an intervention under development. Effects could increase or decrease as development continues and the program scales. Although this study was planned long before the current pandemic, results are particularly timely now that many students are at home under shelter-in-place orders due to COVID-19. The approach taken here is feasible for students at home, with tutors supporting them from a distance. It is also feasible in many other situations where equity could be addressed directly by supporting students via online tutors.

1.1 Macroeconomic summary Economic recovery has consistently outperformed the technical staff’s expectations following a steep decline in activity in the second quarter of 2020. At the same time, total and core inflation rates have fallen and remain at low levels, suggesting that a significant element of the reactivation of Colombia’s economy has been related to recovery in potential GDP. This would support the technical staff’s diagnosis of weak aggregate demand and ample excess capacity. The most recently available data on 2020 growth suggests a contraction in economic activity of 6.8%, lower than estimates from January’s Monetary Policy Report (-7.2%). High-frequency indicators suggest that economic performance was significantly more dynamic than expected in January, despite mobility restrictions and quarantine measures. This has also come amid declines in total and core inflation, the latter of which was below January projections if controlling for certain relative price changes. This suggests that the unexpected strength of recent growth contains elements of demand, and that excess capacity, while significant, could be lower than previously estimated. Nevertheless, uncertainty over the measurement of excess capacity continues to be unusually high and marked both by variations in the way different economic sectors and spending components have been affected by the pandemic, and by uneven price behavior. The size of excess capacity, and in particular the evolution of the pandemic in forthcoming quarters, constitute substantial risks to the macroeconomic forecast presented in this report. Despite the unexpected strength of the recovery, the technical staff continues to project ample excess capacity that is expected to remain on the forecast horizon, alongside core inflation that will likely remain below the target. Domestic demand remains below 2019 levels amid unusually significant uncertainty over the size of excess capacity in the economy. High national unemployment (14.6% for February 2021) reflects a loose labor market, while observed total and core inflation continue to be below 2%. Inflationary pressures from the exchange rate are expected to continue to be low, with relatively little pass-through on inflation. This would be compatible with a negative output gap. Excess productive capacity and the expectation of core inflation below the 3% target on the forecast horizon provide a basis for an expansive monetary policy posture. The technical staff’s assessment of certain shocks and their expected effects on the economy, as well as the presence of several sources of uncertainty and related assumptions about their potential macroeconomic impacts, remain a feature of this report. The coronavirus pandemic, in particular, continues to affect the public health environment, and the reopening of Colombia’s economy remains incomplete. The technical staff’s assessment is that the COVID-19 shock has affected both aggregate demand and supply, but that the impact on demand has been deeper and more persistent. Given this persistence, the central forecast accounts for a gradual tightening of the output gap in the absence of new waves of contagion, and as vaccination campaigns progress. The central forecast continues to include an expected increase of total and core inflation rates in the second quarter of 2021, alongside the lapse of the temporary price relief measures put in place in 2020. Additional COVID-19 outbreaks (of uncertain duration and intensity) represent a significant risk factor that could affect these projections. Additionally, the forecast continues to include an upward trend in sovereign risk premiums, reflected by higher levels of public debt that in the wake of the pandemic are likely to persist on the forecast horizon, even in the context of a fiscal adjustment. At the same time, the projection accounts for the shortterm effects on private domestic demand from a fiscal adjustment along the lines of the one currently being proposed by the national government. This would be compatible with a gradual recovery of private domestic demand in 2022. The size and characteristics of the fiscal adjustment that is ultimately implemented, as well as the corresponding market response, represent another source of forecast uncertainty. Newly available information offers evidence of the potential for significant changes to the macroeconomic scenario, though without altering the general diagnosis described above. The most recent data on inflation, growth, fiscal policy, and international financial conditions suggests a more dynamic economy than previously expected. However, a third wave of the pandemic has delayed the re-opening of Colombia’s economy and brought with it a deceleration in economic activity. Detailed descriptions of these considerations and subsequent changes to the macroeconomic forecast are presented below. The expected annual decline in GDP (-0.3%) in the first quarter of 2021 appears to have been less pronounced than projected in January (-4.8%). Partial closures in January to address a second wave of COVID-19 appear to have had a less significant negative impact on the economy than previously estimated. This is reflected in figures related to mobility, energy demand, industry and retail sales, foreign trade, commercial transactions from selected banks, and the national statistics agency’s (DANE) economic tracking indicator (ISE). Output is now expected to have declined annually in the first quarter by 0.3%. Private consumption likely continued to recover, registering levels somewhat above those from the previous year, while public consumption likely increased significantly. While a recovery in investment in both housing and in other buildings and structures is expected, overall investment levels in this case likely continued to be low, and gross fixed capital formation is expected to continue to show significant annual declines. Imports likely recovered to again outpace exports, though both are expected to register significant annual declines. Economic activity that outpaced projections, an increase in oil prices and other export products, and an expected increase in public spending this year account for the upward revision to the 2021 growth forecast (from 4.6% with a range between 2% and 6% in January, to 6.0% with a range between 3% and 7% in April). As a result, the output gap is expected to be smaller and to tighten more rapidly than projected in the previous report, though it is still expected to remain in negative territory on the forecast horizon. Wide forecast intervals reflect the fact that the future evolution of the COVID-19 pandemic remains a significant source of uncertainty on these projections. The delay in the recovery of economic activity as a result of the resurgence of COVID-19 in the first quarter appears to have been less significant than projected in the January report. The central forecast scenario expects this improved performance to continue in 2021 alongside increased consumer and business confidence. Low real interest rates and an active credit supply would also support this dynamic, and the overall conditions would be expected to spur a recovery in consumption and investment. Increased growth in public spending and public works based on the national government’s spending plan (Plan Financiero del Gobierno) are other factors to consider. Additionally, an expected recovery in global demand and higher projected prices for oil and coffee would further contribute to improved external revenues and would favor investment, in particular in the oil sector. Given the above, the technical staff’s 2021 growth forecast has been revised upward from 4.6% in January (range from 2% to 6%) to 6.0% in April (range from 3% to 7%). These projections account for the potential for the third wave of COVID-19 to have a larger and more persistent effect on the economy than the previous wave, while also supposing that there will not be any additional significant waves of the pandemic and that mobility restrictions will be relaxed as a result. Economic growth in 2022 is expected to be 3%, with a range between 1% and 5%. This figure would be lower than projected in the January report (3.6% with a range between 2% and 6%), due to a higher base of comparison given the upward revision to expected GDP in 2021. This forecast also takes into account the likely effects on private demand of a fiscal adjustment of the size currently being proposed by the national government, and which would come into effect in 2022. Excess in productive capacity is now expected to be lower than estimated in January but continues to be significant and affected by high levels of uncertainty, as reflected in the wide forecast intervals. The possibility of new waves of the virus (of uncertain intensity and duration) represents a significant downward risk to projected GDP growth, and is signaled by the lower limits of the ranges provided in this report. Inflation (1.51%) and inflation excluding food and regulated items (0.94%) declined in March compared to December, continuing below the 3% target. The decline in inflation in this period was below projections, explained in large part by unanticipated increases in the costs of certain foods (3.92%) and regulated items (1.52%). An increase in international food and shipping prices, increased foreign demand for beef, and specific upward pressures on perishable food supplies appear to explain a lower-than-expected deceleration in the consumer price index (CPI) for foods. An unexpected increase in regulated items prices came amid unanticipated increases in international fuel prices, on some utilities rates, and for regulated education prices. The decline in annual inflation excluding food and regulated items between December and March was in line with projections from January, though this included downward pressure from a significant reduction in telecommunications rates due to the imminent entry of a new operator. When controlling for the effects of this relative price change, inflation excluding food and regulated items exceeds levels forecast in the previous report. Within this indicator of core inflation, the CPI for goods (1.05%) accelerated due to a reversion of the effects of the VAT-free day in November, which was largely accounted for in February, and possibly by the transmission of a recent depreciation of the peso on domestic prices for certain items (electric and household appliances). For their part, services prices decelerated and showed the lowest rate of annual growth (0.89%) among the large consumer baskets in the CPI. Within the services basket, the annual change in rental prices continued to decline, while those services that continue to experience the most significant restrictions on returning to normal operations (tourism, cinemas, nightlife, etc.) continued to register significant price declines. As previously mentioned, telephone rates also fell significantly due to increased competition in the market. Total inflation is expected to continue to be affected by ample excesses in productive capacity for the remainder of 2021 and 2022, though less so than projected in January. As a result, convergence to the inflation target is now expected to be somewhat faster than estimated in the previous report, assuming the absence of significant additional outbreaks of COVID-19. The technical staff’s year-end inflation projections for 2021 and 2022 have increased, suggesting figures around 3% due largely to variation in food and regulated items prices. The projection for inflation excluding food and regulated items also increased, but remains below 3%. Price relief measures on indirect taxes implemented in 2020 are expected to lapse in the second quarter of 2021, generating a one-off effect on prices and temporarily affecting inflation excluding food and regulated items. However, indexation to low levels of past inflation, weak demand, and ample excess productive capacity are expected to keep core inflation below the target, near 2.3% at the end of 2021 (previously 2.1%). The reversion in 2021 of the effects of some price relief measures on utility rates from 2020 should lead to an increase in the CPI for regulated items in the second half of this year. Annual price changes are now expected to be higher than estimated in the January report due to an increased expected path for fuel prices and unanticipated increases in regulated education prices. The projection for the CPI for foods has increased compared to the previous report, taking into account certain factors that were not anticipated in January (a less favorable agricultural cycle, increased pressure from international prices, and transport costs). Given the above, year-end annual inflation for 2021 and 2022 is now expected to be 3% and 2.8%, respectively, which would be above projections from January (2.3% and 2,7%). For its part, expected inflation based on analyst surveys suggests year-end inflation in 2021 and 2022 of 2.8% and 3.1%, respectively. There remains significant uncertainty surrounding the inflation forecasts included in this report due to several factors: 1) the evolution of the pandemic; 2) the difficulty in evaluating the size and persistence of excess productive capacity; 3) the timing and manner in which price relief measures will lapse; and 4) the future behavior of food prices. Projected 2021 growth in foreign demand (4.4% to 5.2%) and the supposed average oil price (USD 53 to USD 61 per Brent benchmark barrel) were both revised upward. An increase in long-term international interest rates has been reflected in a depreciation of the peso and could result in relatively tighter external financial conditions for emerging market economies, including Colombia. Average growth among Colombia’s trade partners was greater than expected in the fourth quarter of 2020. This, together with a sizable fiscal stimulus approved in the United States and the onset of a massive global vaccination campaign, largely explains the projected increase in foreign demand growth in 2021. The resilience of the goods market in the face of global crisis and an expected normalization in international trade are additional factors. These considerations and the expected continuation of a gradual reduction of mobility restrictions abroad suggest that Colombia’s trade partners could grow on average by 5.2% in 2021 and around 3.4% in 2022. The improved prospects for global economic growth have led to an increase in current and expected oil prices. Production interruptions due to a heavy winter, reduced inventories, and increased supply restrictions instituted by producing countries have also contributed to the increase. Meanwhile, market forecasts and recent Federal Reserve pronouncements suggest that the benchmark interest rate in the U.S. will remain stable for the next two years. Nevertheless, a significant increase in public spending in the country has fostered expectations for greater growth and inflation, as well as increased uncertainty over the moment in which a normalization of monetary policy might begin. This has been reflected in an increase in long-term interest rates. In this context, emerging market economies in the region, including Colombia, have registered increases in sovereign risk premiums and long-term domestic interest rates, and a depreciation of local currencies against the dollar. Recent outbreaks of COVID-19 in several of these economies; limits on vaccine supply and the slow pace of immunization campaigns in some countries; a significant increase in public debt; and tensions between the United States and China, among other factors, all add to a high level of uncertainty surrounding interest rate spreads, external financing conditions, and the future performance of risk premiums. The impact that this environment could have on the exchange rate and on domestic financing conditions represent risks to the macroeconomic and monetary policy forecasts. Domestic financial conditions continue to favor recovery in economic activity. The transmission of reductions to the policy interest rate on credit rates has been significant. The banking portfolio continues to recover amid circumstances that have affected both the supply and demand for loans, and in which some credit risks have materialized. Preferential and ordinary commercial interest rates have fallen to a similar degree as the benchmark interest rate. As is generally the case, this transmission has come at a slower pace for consumer credit rates, and has been further delayed in the case of mortgage rates. Commercial credit levels stabilized above pre-pandemic levels in March, following an increase resulting from significant liquidity requirements for businesses in the second quarter of 2020. The consumer credit portfolio continued to recover and has now surpassed February 2020 levels, though overall growth in the portfolio remains low. At the same time, portfolio projections and default indicators have increased, and credit establishment earnings have come down. Despite this, credit disbursements continue to recover and solvency indicators remain well above regulatory minimums. 1.2 Monetary policy decision In its meetings in March and April the BDBR left the benchmark interest rate unchanged at 1.75%.

The Kyrgyz Republic has overcome some complex challenges to now have one of the most open economies in Central Asia. The country has improved its per capita income and living standards and is on the threshold of becoming a lower-middle-income economy. Looking ahead, the main challenge for the Kyrgyz Republic is to build on these successes to stimulate stronger, more broad-based economic growth. This book identifies the economic and governance reforms needed within key sectors to drive this growth. It provides policy suggestions to enhance the country’s trade, agriculture, tourism, finance, information and communications technology, energy, transport and logistics, and human capital.

En los primeros meses de 2021 la recuperación que venía registrando el empleo total desde mediados de 2020 se detuvo. Buena parte de este fenómeno habría estado relacionado con las olas de contagio de Covid-19, que involucraron tanto respuestas de autocuidado por parte de los individuos como la implementación de medidas de restricción a la movilidad en varias ciudades para mitigar el contagio. Ambas circunstancias produjeron fuertes caídas mensuales del empleo en enero y en abril. En mayo la prolongación de la tercera ola de contagios y la situación de orden público por la que atravesó el país volvieron a limitar la recuperación del número de ocupados. En medio de este panorama, un hecho favorable lo registró el empleo asalariado y formal que, a diferencia de su contraparte no asalariada e informal, presentó crecimientos sutiles. La expansión en este segmento, que suele ser menos rápida por los mayores costos de contratación, se observa no solo en la encuesta de hogares sino también en los registros administrativos, y está favorecida por un mayor número de vacantes disponibles y tasas de separación en niveles muy bajos. Por ramas de actividad el comportamiento del empleo ha sido heterogéneo, y en la mayoría de los sectores se registran recuperaciones del empleo más lentas que las de la actividad económica, lo que ha implicado, al menos en términos contables, aumentos de la productividad laboral. Por otro lado, la salida de la inactividad se detuvo, por lo que la participación laboral se mantuvo relativamente estable en los últimos meses. Así las cosas, la incipiente recuperación del empleo y la estabilidad de la oferta laboral conllevaron a que las tasas de desempleo (TD) de mayo se ubicaran en niveles similares a lo observado en lo corrido de 2021: 15,2% y 16,5% en el total nacional y las trece ciudades, respectivamente. Al descomponer dichas tasas por grupos poblacionales se observan aún importantes disparidades, en particular en las mujeres y los jóvenes. Además, se registran tendencias ascendentes en el número de desempleados que permanecen varios meses en el desempleo y de los inactivos que transitan al desempleo. Finalmente, por el lado de los ingresos, los del segmento no asalariado urbano, los más golpeados durante la pandemia, se han seguido recuperando, y sus horas trabajadas ya casi se encuentran en los niveles previos a la pandemia. Dado lo anterior, en este reporte se estima que la TD empezará a mostrar reducciones en lo que queda de 2021, aunque moderadas. Se espera que la TD nacional se ubique en promedio en 2021 entre el 13,7% y 15%, con 14,4% como valor central, en cuyo caso cerraría el año alrededor del 13,8%. Las estimaciones de la TD de largo plazo consistente con una inflación estable (Nairu) sugieren una brecha de la TD positiva de alrededor de 2,3 puntos porcentuales (pp) para 2021, por lo que la holgura del mercado laboral continuaría, presionando a la baja la inflación vía costos salariales. Como es usual, este reporte se divide en dos secciones. En la primera se examinan en detalle los principales hechos coyunturales del mercado laboral. En la segunda se estudia la evolución reciente del mercado laboral de los jóvenes, los cuales han sido protagonistas de las manifestaciones registradas en el país durante mayo, y se analizan los efectos que han tenido programas para dinamizar la creación de empleo juvenil implementados en el pasado. En particular, se estiman los efectos de la Ley del Primer Empleo de 2010 y se recopilan los resultados encontrados en la literatura sobre el programa Jóvenes en Acción. Se encuentra que ambas políticas son complementarias y efectivas en reducir el desempleo juvenil, pero conllevan costos fiscales, los cuales requieren estrategias de financiación sostenibles. Adicionalmente, a partir de la estimación realizada, se efectúa una cuantificación aproximada de los efectos de eliminar los impuestos de nómina para los jóvenes. Este ejercicio proporciona un punto de referencia para diagnosticar los posibles efectos que el Decreto 668 de 2021, promulgado recientemente por el Gobierno, y consistente en un subsidio temporal a la nómina de nuevos empleados jóvenes, tendría sobre el desempleo juvenil.